Nucleic acid molecules encoding proteins which impart the adhesion of neisseria cells to human cells

ABSTRACT

Described are nucleic acid molecules encoding proteins mediating the adhesion of bacteria of the genus Neisseria to human cells. Also described are the proteins encoded by these nucleic acid molecules and antibodies directed against them. Furthermore, pharmaceutical compositions, vaccines and diagnostic compositions containing the nucleic acid molecules, proteins and/or antibodies are described.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No., PCT/EP96/04092, which has anInternational filing date of Sep. 18, 1996, which designated the UnitedStates of America, the entire contents of which are hereby incorporatedby reference.

The present invention relates to nucleic acid molecules from bacteria ofthe genus Neisseria encoding proteins mediating the adhesion ofNeisseria cells to human cells. Furthermore, the present inventionrelates to the proteins encoded by these nucleic acid molecules and toantibodies directed against them. The present invention further relatesto pharmaceutical compositions, vaccines and diagnostic compositionscontaining said nucleic acid molecules, proteins and/or antibodies.

To the genus Neisseria (gram-negative cocci) belong a number ofbacterial species which, being saprophytes, populate the upper humanrespiratory tract. Apart from commensal species (e.g.: N. sicca) andopportunistically pathogenic species (e.g.: N. lactamica), two Neisseriaspecies are known which clearly possess human-pathogenic properties. Oneof the species is N. gonorrhoeae, the pathogen of the venereal diseasegonorrhea, which exclusively occurs in humans, and N. meningitidis, thepathogen of the bacterial epidemic meningitis. In both cases theetiology, that is the causal connection between the development of theclinical picture and the population by bacteria from said species hasmeanwhile been substantiated.

The purulent meningitis (Meningitidis cerebrospinalis epidemica) causedby N. meningitidis (“meningococcus”), which usually is epidemical, is asystemic invasive infection of the human meninx and spinal meninx.Occasionally, hemorrhagic exanthema at the trunk or concomitant diseasescaused by Herpes simplex can be observed in addition. The pathogen canappear in the form of several serotypes, which are distinguishable bymeans of agglutination assays with immune sera. The main groups differremarkably, and their prevalence differs with regard to when and wherethey appear. Meningococcus meningitidis has up to now occurred in largenumbers every 8 to 12 years with the increased prevalence lastingbetween 4 to 6 years. While serovar B meningococci brought on 50% to 55%of the recent diseases to the civilian population as well as to themilitary personnel in the United States, most epidemic diseases in theUnited States during the first half of the century were caused byserovar A meningococci.

The clinical picture caused by N. gonorrhoeae usually is an infectionlocalized to the mucous membranes, in most cases of the urogenital tract(gonorrhea), more rarely of the conjunctiva (conjunctivis gonorrhoeae,gonoblennorrhoe), which is acquired by new born children perinatally, byadults usually unilaterally by smear infection. In very rare casesbacteremia and sepsis occur after hematogenous dissemination. As aconsequence, exanthema with hemorrhagic pustules, diseases from therheumatic Formenkreis, arthritis gonorrhoica and/or endocarditis canoccur.

Usually, the diseases caused by N. gonorrhoeae and N. meningitidis aretreated with antibiotics. More and more, however, the bacteria arebecoming resistant to single or groups of the antibiotics used so thatthe therapy method that has nearly exclusively been used up to now willmost likely not be successful in the long run. Therefore, it isdesirable and urgent that alternative therapy methods, preferablypreventive ones, be developed.

Neisseria gonorrhoeae and N. meningitidis exclusively occur in humans.They have adapted to the host organism and show a number of propertiesthat are able to make the defense mechanisms of the host ineffective.Therefore, up to now there is no vaccine available that preventsgonorrhea. This is to a limited extent also true for meningococcusmeningitidis. Even though the disease has recently been caused mainly bybacteria of the same serovar, group B, no effective vaccine againstmeningococci of group B has existed up to now. Vaccines against otherserovars only offer partial protection and are not unproblematic from animmunological point of view. The reason for the failure of the immunedefense is, inter alia, the antigen variation of the pathogens, which inthe case of the pathogenic Neisseria is particularly developed. However,a limitation of the free development of the antigen variation seems tobe necessary where the functional region has to be sterically maintainedin order to guarantee the interaction with conserved and constantstructures of the host receptors. This requirement especially applies tothe adhesins that serve for adhering to the host cell. Only if thefunctional region that is involved in the physical interaction is keptconstant, the interaction with the receptor of the host cell ispossible. This region should be excluded from antigen variation to alarge extent and is therefore a suitable starting-point for thedevelopment of a new therapy method.

The initial phase of infections usually is the stable adhesion of thepathogens to the host tissue. By interactions between structures of thecell surface of the pathogens and the cell surface of the host cell amechanically stable linkage is formed that allows the bacteria to stayon the tissue of the host (colonization) and to subsequently propagatelocally. The adhesion to the host cell can be divided into two phaseswith different structures being involved in the interaction.

In the first phase of adhesion a contact between host cell and pathogenis mediated. Often cell appendage organelles, the so-called pili, areinvolved in mediating the contact. These cell organelles, which are alsocalled fimbriae or fibrils, are few to several fine filamentous rigid orflexible appendages of the bacterial cell, which can be several times aslong as the cell diameter. Therefore, there is no contact between thecell walls of pathogen and host cell in the pilus mediated adhesion. Themajority of the known pili are heteropolymeric structures consisting ofseveral components. The main subunit, which usually is present in manycopies, fulfills the structural function, that is the frameworkfunction, whereas the actual adhesion function is fulfilled by sidecomponents, which usually are present in few copies.

A further form of adherence is the adhesion of pathogens to the hostcells without the contribution of pili (pilus independent adherence,pia). In this case, the pathogen and the host cell are approaching eachother, and finally the cell walls directly touch. This adhesion andstabilization of the contact between the cells takes place with thecontribution of adhesines that are located in the bacterial cell wall.As a result of the direct contact between the cells, a signal is finallytransmitted that initiates the pathogen induced phagocytosis and startsthe invasion process into the target cell. The pia form of adherence canautonomically effect the adhesion of pathogens, for example in the caseof pathogens lacking pili. It can, however, also act as the second phaseof adhesion, that is as the consecutive reaction after pilus mediatedadhesion, and stabilize the contact between the cells. The adhesinesthat are involved in the pilus independent adhesion can but do notnecessarily have to show different binding specifities from those thatare involved in pilus dependent adhesion.

In the context of the invention the bacterial structures that areinvolved in the adhesion will in the following be called adhesines,those of the host cells will be called receptors. If there is no contactbetween adhesin and receptor, “defense mechanisms” of the host, such asfibrillation of the epithelia, mucus secretion, mass flow of body fluidsand the like, eliminate the pathogens. The development of an infectionis, therefore, prevented from the very beginning. Thus, a disturbance ofthe adhesion of the pathogens by means of inhibiting the interactionbetween adhesin and receptor of the target cell represents a veryeffective approach for preventing and treating infections. Suchtherapeutically effective approaches comprise the production ofantibodies specifically blocking the adhesin function, either by activeimmunization (vaccination) or by administration of antibodies alreadyexisting (passive immunization). The adhesin receptor binding can, inthe same way, be inhibited by means of passive administration of bothreceptor analogous and adhesin analogous substances. These substancescompetitively bind to the corresponding partner structures, therebyblocking their involvement in productive interactions. In the context ofthe invention such substances are called inhibitors.

The approaches using pilin, the main component of the pilus thatfulfills the structural function, in order to develop a broadlyeffective vaccine effectively blocking the adhesion of pathogenicNeisseria have failed so far. The reason probably is that (i) pilinitself has no adhesin function and (ii) pilin possesses an especiallydistinct intra- and interstem specific antigenic variation. Since bothlimitations, as described above, do not apply to adhesins, the use of anadhesin as a vaccine is more promising.

The technical problem of the present invention therefore is to provideproteins and DNA molecules encoding them that serve as adhesionstructures for Neisseria species or contribute to the development ofsuch structures.

This problem is solved by providing the embodiments described in theclaims.

Therefore, the present invention relates to nucleic acid moleculescontaining the nucleotide sequence described in SEQ ID NO: 1 or partsthereof with these nucleic acid molecules comprising one or more openreading frames encoding proteins or biologically active fragmentsthereof from bacteria of the genus Neisseria that mediate the adhesionof Neisseria cells to human cells. The term “reading frame” in thiscontext is used synonymously with the term “coding region”.

The subject matter of the invention also relates to nucleic acidmolecules that basically show the nucleotide sequence described in SEQID NO: 1 but whereby the nucleotide sequences of the open reading framesdeviate from those described in SEQ ID NO: 1 due to the degeneration ofthe genetic code. Preferably, the open reading frames of those nucleicacid molecules have nucleotide sequences encoding proteins with one ofthe amino acid sequences described in SEQ ID NO. 1. The subject matterof the invention further relates to nucleic acid molecules hybridizingto the nucleic acid molecules described above and comprising codingregions encoding proteins that mediate the adhesion of Neisseria cellsto human cells.

In the context of the present invention the term “hybridization” is usedas described in Sambrook et al. (Molecular Cloning, A Laboratory Manual;Cold Spring Harbor Laboratory Press (1989), 1.101 to 1.104). Preferably,this term has the meaning of hybridization under stringent conditions.In particular, it has the meaning of a hybridization that still shows apositive hybridization signal after being washed for 1 h with 1×SSC and0.1% SDS, preferably with 0.2×SSC and 0.1% SDS, at 55° C., preferably at62° C. and most preferably at 68° C.

In a preferred embodiment the nucleic acid molecule of the inventionoriginates from a pathogenic Neisseria species, in particular fromNeisseria gonorrhoea or Neisseria meningitidis.

The term “nucleic acid molecule” as used here according to the inventionrelates to the polymeric form of nucleotides of any length, either asribonucleotides or as desoxyribonucleotides. The term only relates tothe primary structure of the molecule. In this sense, it comprises DNAand RNA molecules, in single- or double-stranded form. The DNA caneither be cDNA or genomic DNA. The term further comprises thenon-modified form as well as scientifically known modifications, e.g.,methylation, capping, base substitution with natural or syntheticanalogues, internucleotide modifications with uncharged compounds (e.g.,methyl phosphate, phosphoamidate, carbamate, phosphotriester and thelike) or with charged compounds (e.g., phosphorothioate,phosphorodithioate and the like) or with binding components such asproteins and peptides (e.g., nucleases, toxins, antibodies,poly-L-lysine, and the like). The term also comprises forms withintercalating substances (e.g., acridin, psoralen, and the like),chelators (e.g., with metals, radioactive metals or oxidizing metals andthe like), with alkylating agents and finally with modified bonds (e.g.,alpha anomeric nucleic acids, and the like).

The invention also relates to vectors containing a nucleic acid moleculeof the invention. The vector can be any prokaryotic or eukaryoticvector. Examples of prokaryotic vectors are chromosomal vectors, such asbacteriophages (e.g., bacteriophage lambda, P1), and extrachromosomalvectors, such as plasmids with circular plasmids being particularlypreferred. Suitable prokaryotic vectors are, for example, described inSambrook et al. (see above), chapters 1 to 4. The vector according tothe invention can also be a eukaryotic vector, for example a yeastvector or a vector suitable for higher cells (e.g., a plasmid vector, aviral vector, a plant vector, and the like). Examples of such vectorsare also described in Sambrook et al. (see above, chapter 16). A vectorcontaining a nucleic acid molecule of the invention is, for example,plasmid pES25 (contained in the E. coli strain H 2560 (DSM 10257)). TheE. coli strain H 2560 was deposited on Sep. 18, 1995 with DeutscheSammlung von Mikroorganismen (DSM) [German collection of microorganisms]in Brunswick, Federal Republic of Germany, as international recognizeddepositary authority in accordance with the stipulations of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure under accession number DSM 10257.

The invention furthermore relates to host cells containing a vector asdescribed above or being genetically manipulated with a nucleic acidmolecule as described above. The term “host cell” in the context of thisinvention comprises both prokaryotic and eukaryotic host cells.Prokaryotic cells are preferred, particularly gram-negative prokaryoticcells, in particular E. coli cells. Suitable eukaryotic host cells are,for example, fungal cells (e.g., yeast cells), animal or plant cells.

The nucleotide sequence described in SEQ ID NO: 1 comprises three openreading frames. They represent an operon forming a functional unity. Thethree open reading frames called orfI, orfA and orfB encode threeproteins that in the context of this invention are called OrfI, OrfA andOrfB. These sequences are responsible for the expression of a protein inNeisseria cells, in particular of the protein OrfA, which is involved inthe adhesion of Neisseria cells to human cells. The proteins OrfI andOrfB obviously possess a regulatory function or a function as factorsthat are able to influence the functionality of OrfA.

This nucleic acid molecule therefore represents a region of theNeisseria genome that encodes proteins having the adhesin function ofNeisseria cells.

The present invention further relates to nucleic acid molecules encodinga lipoprotein or biologically active fragments thereof from bacteria ofthe genus Neisseria having the amino acid sequence as described in SEQID NO: 6. In a preferred embodiment the invention relates to nucleicacid molecules encoding a protein having the amino acid sequence fromthe amino acid residue 19 to the amino acid residue 320 of the aminoacid sequence as described in SEQ ID NO: 6. Such nucleic acid moleculespreferably have the nucleotide sequence described in SEQ ID NO: 6, inparticular the nucleotide sequence from nucleotide 189 to nucleotide1095 of the sequence described in SEQ ID NO: 6.

The subject matter of the invention also relates to nucleic acidmolecules encoding a lipoprotein from bacteria of the genus Neisseriawhereby their nucleotide sequence deviates from the nucleic acidmolecules described above due to the degeneration of the genetic code.

Furthermore, the present invention relates to nucleic acid moleculesencoding a lipoprotein from bacteria of the genus Neisseria andhybridize to one of the nucleic acid molecules described above (for thedefinition of the term “hybridization” see above).

The subject matter of the invention also relates to fragments,derivatives and allelic variants of the nucleic acid molecules describedabove that encode the lipoprotein described above. Fragments areunderstood to be parts of the nucleic acid molecules that are longenough to encode the protein described. The term derivative in thiscontext means that the nucleotide sequences of these molecules differ atone or more positions from the sequences of the nucleic acid moleculesdescribed above and that they show a high level of homology to thesenucleotide sequences. Homology means a sequence identity of at least40%, in particular an identity of at least 60%, preferably of more than80% and particularly preferred of more than 90%. The deviations to thenucleic acid molecules described above can be caused by deletion,substitution, insertion or recombination.

Homology further means that there is a functional and/or structuralequivalence between the corresponding nucleic acid molecules or theproteins encoded by them. The nucleic acid molecules that are homologousto those described above and that represent derivatives of these nucleicacid molecules usually are variants of these molecules displayingmodifications that have the same biological function. They can benaturally occurring variants, for example sequences from otherorganisms, or mutations, which either occur naturally or that have beenintroduced by means of specific mutagenesis. Furthermore, the variantscan be synthetically produced sequences.

The allelic variants can be both naturally occurring variants orvariants that were synthetically produced or that were produced byrecombinant DNA techniques.

The proteins encoded by the various variants of the nucleic acidmolecules according to the invention show certain commoncharacteristics, for example enzyme activity, molecular weight,immunological reactivity, conformation etc., as well as physicalproperties such as the electorphoretic mobility, chromatographicbehavior, sedimentation coefficients, solubility, spectroscopicproperties, stability, pH optimum, temperature optimum etc.

Preferably, the proteins encoded by the nucleic acid molecules accordingto the invention show a homology of 80%, particularly preferred of morethan 90% to the nucleotide sequence described in SEQ ID NO: 6.

The nucleic acid molecules described above encode a lipoprotein frombacteria of the genus Neisseria. This protein is called OrfA in thecontext of the present invention. This protein is, according toexperimental data, located on the cell surface of Neisseria cells, inparticular on the outer membrane. The protein preferably has a molecularweight of about 36 kd if it is analyzed in the T7 expression system.

Furthermore, this protein possesses a biological activity that mediatesthe adhesion of Neisseria cells to human cells. This is in particularlymade because this protein forms a complex with the protein fromNeisseria known as PilC. The adhesion preferably takes place on humanepithelial cells.

Furthermore, the invention relates to vectors containing nucleic acidmolecules described above. Examples of such vectors have already beendescribed above.

In a preferred embodiment the DNA molecules according to the inventionare linked in such vectors with regulatory DNA elements that make theexpression of the protein in prokaryotic or eukaryotic cells possible.Examples thereof are in the context of this invention promoters,operators, enhancers and the like.

Furthermore, the invention relates to host cells that contain vectorsaccording to the invention described above or that have been geneticallymanipulated with the nucleic acid molecules described above. Geneticallymanipulated means that such a molecule has been introduced into the hostcell or in a precursor cell by means of (gene) technological methods.Again, the above-described host cells are suitable.

The invention also relates to methods for the production of thedescribed lipoprotein or a biologically active fragment thereof wherebythe host cells described above are cultivated under conditions thatallow the expression of the protein and the protein is isolated from thecells and/or the culture supernatant.

The invention also relates to proteins encoded by one of the nucleicacid molecules described above, as well as to biologically activefragments thereof as well as to proteins available by the methoddescribed above. In particular, the invention relates to proteins havingamino acid sequences that immunologically cross-react with the describedproteins. The term “protein” comprises in the context of the presentinvention also naturally occurring variants or modifications orfragments or synthetically produced modifications, variants or fragmentswith the corresponding biological activity. Derived or recombinantproteins do not necessarily have to be biologically translated from thenucleotide sequence. They can be produced in any way, including chemicalsynthesis, in vitro synthesis by means of an expression system or byisolation from organisms. Proteins according to the invention cancontain one or more amino acid analogues or amino acids not naturallyoccurring. Also, modifications (e.g., glycosylation, and the like) orlabeling (e.g., biotinylation) according to the scientific knowledge canbe contained.

The fragments preferably have a length of at least 3 to 5 amino acids,particularly preferred of 8 to 10 amino acids and in particularpreferred of 11 to 15 amino acids. This is also true for the proteinsaccording to the invention described below.

The lipoprotein OrfA according to the invention can be purified, forexample, by a method that is based on the interaction of this proteinwith the PilC protein from Neisseria gonorrhoeae. It is preferablypurified from homogenates of cells expressing this protein by means ofchromatography matrices containing immobilized PilC protein. The proteincan then be selectively eluted using its affinity to PilC and producedin essentially pure form.

The proteins according to the invention or fragments thereof can be usedas immunogens for the production of antibodies. Therefore, the presentinvention also relates to antibodies that are directed against a proteinaccording to the invention or a fragment thereof. The antibodies can beboth polyclonal and monoclonal. Methods for the production of suchantibodies are known to the skilled person.

In a preferred embodiment such antibodies are directed against epitopesof the protein according to the invention or fragments thereof that areimportant for the adherence and for the interaction with PilC.

The antibodies according to the invention can be, for example, producedby introducing the nucleic acid sequences according to the inventiondescribed above into hosts by in vivo transfection. Thereby, the proteinor a fragment thereof is expressed in the host and the antibodiesdirected against them are induced (nucleic acid vaccination). This isalso the case with the antibodies described below.

The present invention further relates to nucleic acid molecules having alength of at least 12 nucleotides and specifically hybridizing to thenucleic acid molecule described above. Preferably, such nucleic acidmolecules have a length of at least 15 nucleotides, particularlypreferably of 20 nucleotides. Such molecules are, for example, suitableas primers for in vitro amplification, for example by polymerase chainreaction (PCR), or suitable for diagnostic purposes, that is forspecifically identifying the nucleic acid molecules of the invention insamples.

The invention further relates to pharmaceutical compositions containinga nucleic acid molecule according to the invention described above, aprotein, a biologically active fragment thereof and/or an antibodyaccording to the invention described above. In the context of thepresent invention such pharmaceutical compositions can contain the usualpharmaceutical adjuvants, diluents, additives and/or carriers. Theinvention also relates to vaccines containing the nucleic acid moleculesdescribed above, proteins, biologically active fragments thereof and/orantibodies.

In a further aspect the present invention relates to diagnosticcompositions containing the nucleic acid molecules according to theinvention described above, proteins, biologically active fragmentsthereof and/or antibodies.

A further aspect of the present invention relates to receptors andsubstances having receptor function, interacting as ligands with theadhesin according to the invention, the OrfA-PilC complex. Suchsubstances can be identified as competitive inhibitors of the adherencefunction due to their interaction with the OrfA-PilC complex. They canbe surface components of human cells, particularly preferred surfacecomponents of human epithelial cells or chemical substances of anyorigin.

Finally, the present invention relates to inhibitors that influence theinteraction between the OrfA-PilC adhesin complex and its receptors.Enclosed are all substances according to the invention that influencethe interaction between the OrfA-PilC adhesin and its cellular receptorand therefore disturb the adherence. In a particularly preferredembodiment substances that irreversibly bind to the adhesin complex suchas receptor analogues are encompassed.

Finally, the present invention relates to pharmaceutical compositionscontaining as an agent

(a) a receptor according to the invention;

(b) a receptor analogue according to the invention; and/or

(c) an inhibitor according to the invention,

optionally together with the usual pharmaceutical adjuvants, diluents,additives and carriers.

The pharmaceutical compositions described in the context of the presentinvention can be used for identifying and characterizing a bacterialsample not yet known as pathogenic Neisseria spc. and for diagnosing aNeisseria infection.

On the polynucleotide level, preferably hybridization probes are usedcontaining the nucleotide sequences of the invention that are specificfor one of the orf-gene regions or nucleotide sequences of the inventionfrom one of the orf gene regions are used as primers for the PCRamplification of the genomic DNA region to be identified that isspecific for pathogenic Neisseria.

On the polypeptide level diagnosis is preferably performed with the helpof antibodies of the invention or, in the case of antibody screeningtests, with the help of immunogenic proteins of the invention orfragments thereof.

Receptors, receptor analogous substances and inhibitors of theinteraction between the OrfA of the invention and the correspondingreceptors of the host cells can be used as therapeutics for infectionsat an early stage or if an infection is suspected. By stronglyinhibiting the adherence, the adhesion of the pathogens to theepithelial host cells can be prevented so that by the usual defensemechanisms, such as ciliary movement of the epithelial cells, mucussecretion, mass flow of body fluids and the like, the pathogens can beeliminated.

Finally, the pharmaceutical compositions of the invention can be usedfor preventing or fighting Neisseria infections. Preferably, forpreventive applications the proteins of the invention or fragmentsthereof are used for the production of a vaccine for activeimmunization, or antibodies of the invention are used for the productionof a passive vaccine applicable as a therapeutic. The applicationsdescribed above also apply to the pharmaceutical compositions anddiagnostic compositions described below.

The subject matter of the invention further relates to nucleic acidmolecules encoding a protein or a biologically active fragment thereoffrom bacteria of the genus Neisseria having the amino acid sequencedescribed in SEQ ID NO: 8. Such nucleic acid molecules preferably havethe nucleotide sequence described in SEQ ID NO: 8, in particular the oneof the described coding region. The invention also relates to nucleicacid molecules the sequence of which deviates from the sequences of themolecules mentioned above due to the degeneration of the genetic code.Also nucleic acid molecules are the subject matter of the invention thathybridize to the nucleic acid molecules mentioned above (for thedefinition of the term “hybridization” see above). For the possiblevariants of the nucleic acid molecules the same is true what has alreadybeen described in connection with the nucleic acid molecules encodingOrfA.

The invention also relates to vectors containing the described nucleicacid molecules, in particular those in which they are linked toregulatory DNA elements for the expression in prokaryotic or eukaryoticcells, as well as to host cells that contain such vectors or that aregenetically manipulated with the described nucleic acid molecules.

The invention also relates to proteins encoded by the nucleic acidmolecules described above and to proteins containing amino acidsequences that immunologically cross-react with the amino acid sequencedepicted in SEQ ID NO: 8 or fragments thereof. In the context of thisinvention they are called OrfI proteins. The protein from Neisseriagonorrhoeae having the amino acid sequence depicted in Seq ID No. 8shows in the T7 expression system an apparent molecular weight of about18 kd. A homology to presently known proteins could not be shown.Experimental data indicate that the protein is located intracellularlyand possibly has a regulatory function.

This protein can be produced by a method in which a host cell describedabove is cultivated under conditions allowing the expression of theprotein and in which the protein is obtained from the cells and/or theculture supernatant. Therefore, the invention also relates to proteinsobtainable by such a method.

The invention also relates to antibodies against a protein describedabove or a fragment thereof as well as to nucleic acid molecules havinga length of at least 12 nucleotides and specifically hybridizing to anucleic acid molecule described above. Preferably, the molecules have alength of more than 15 nucleotides and particularly preferably of morethan 20 nucleotides.

The invention further relates to pharmaceutical compositions containinga nucleic acid molecule, protein, biologically active fragment thereofand/or an antibody described above and, optionally, a pharmaceuticallyacceptable carrier.

The invention further relates to diagnostic compositions containing thenucleic acid molecules, proteins, biologically active fragments thereofand/or antibodies described above.

The subject matter of the invention further relates to nucleic acidmolecules encoding a protein or a biologically active fragment thereoffrom bacteria of the genus Neisseria that has the amino acid sequencedepicted in SEQ ID NO: 10. Such nucleic acid molecules preferably havethe nucleotide sequence depicted in SEQ ID NO: 10, in particular the oneof the indicated coding region. The invention also relates to nucleicacid molecules the sequences of which deviate from the nucleotidesequence of the above-mentioned molecules due to the degeneration of thegenetic code. Furthermore, the subject matter of the invention alsorelates to nucleic acid molecules hybridizing to the above-mentionednucleic acid molecules (for the definition of the term “hybridization”see above). The same applies to possible variants of the nucleic acidmolecules as has already been described in connection with the nucleicacid molecules encoding OrfA.

In a preferred embodiment the above-described nucleic acid moleculesencode a protein that is able to form a complex with the protein PilCand therefore shows an ability of adherence to human cells.

The invention also relates to vectors containing the described nucleicacid molecules, in particular those in which they are linked toregulatory DNA elements for the expression in prokaryotic or eukaryoticcells, as well as to host cells that contain such vectors or that havebeen genetically manipulated with the above-described nucleic acidmolecules.

The invention also relates to proteins encoded by the above-describednucleic acid molecules and to proteins containing the amino acidsequences that immunologically cross-react with the amino acid sequencedepicted in SEQ ID NO: 10 or parts thereof. These are called OrfB in thecontext of the present invention. The protein from Neisseria gonorrhoeaehaving the amino acid sequence depicted in SEQ ID NO: 10 shows in the T7expression system an apparent molecular weight of about 57 kd. Ahomology to presently known proteins could not be shown. Experimentaldata indicate that the protein is, like OrfA, located at the cellsurface and is accessible from the outside. Furthermore, it obviouslyalso possesses the ability to form a complex with the protein PilC andto induce either alone or in combination with OrfA the adhesion to humancells.

This protein can be produced by a method in which an above-describedhost cell is cultivated under conditions allowing the expression of theprotein and in which the protein is obtained from the cells and/or theculture supernatant. Therefore, the invention also relates to proteinsobtainable by such a method.

The invention also relates to antibodies against an above-describedprotein or fragment thereof, as well as to nucleic acid molecules havinga length of at least 12 nucleotides and specifically hybridizing to anabove-described nucleic acid molecule. Preferably, such molecules have alength of more than 15 nucleotides and particularly preferred of morethan 20 nucleotides.

Furthermore, the invention relates to pharmaceutical compositionscontaining an above-described nucleic acid molecule, protein,biologically active fragment thereof and/or antibody and, optionally,pharmaceutically acceptable carriers.

The subject matter of the invention further relates to diagnosticcompositions containing the above-described nucleic acid molecules,proteins, fragments thereof and/or antibodies.

Illustration of the figures and the sequence protocols:

FIG. 1 schematically shows the construction of the plasmid pES25.

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 1) of the orf generegion, starting from position 1 at the modified BgII cleavage site andending with position 3260, the last nucleotide of the HindIII cleavagesite. Restriction cleavage sites, ribosome binding sites (Shine-Dalgarnosequences) and promoter sequences (−35 and −10 regions) are labeled.

SEQ ID No. 1 further shows the amino acid sequences of the proteinsOrfI, OrfA and OrfB encoded by the orf gene region. The amino acids ofthe lipoprotein signal sequence of OrfA are written in italic, thecleavage sites of the lipoprotein signal peptidase II is labeled withthe tip of an arrow. The amino acid cysteine that represents the aminoterminal of the processed OrfA lipoprotein and is modified to glycerylcysteine with fatty acid is marked with a circle. The first seven aminoacids of OrfB that are similar to a typeIV-pilin-signal sequence arewritten in bold. The labeling between amino acids 7 and 8 and between 11and 12 characterize potential cleavage sites analogous to the processingof the typeIV-pilin.

SEQ ID NO: 6 shows the nucleotide sequence of the gene region encodingOrfA as well as flanking sequences. The amino acid sequence of OrfA isdepicted, too.

SEQ ID NO: 8 shows the nucleotide sequence of the gene region encodingOrfI as well as flanking sequences. The amino acid sequence of OrfI isdepicted, too.

SEQ ID NO: 10 shows the nucleotide sequence of the gene region encodingOrfB as well as flanking sequences. The amino acid sequence of OrfB isdepicted, too.

The examples illustrate the invention.

EXAMPLES Example 1 Method for the Isolation of the Lipoprotein AdhesinOrfA

During the chromatographic purification of the PilC protein a decisiveobservation with regard to the identification of the new adhesin ofNeisseria gonorrhoeae of the invention was made. A recombinant PilCprotein was used that was amplified by an oligo-histidine region withsix histidine residues (His₆-tag) in order to make the chromatographicpurification easier (Rudel et al., Nature 373, 357-359, 1995). Theamplification of the protein by the histidine hexapeptide makes theselective binding to a nickel-nitrilotriacetate-agarose matrix (Ni-NTAmatrix) possible. After the cell wall fraction produced from cultures ofa pilus-free PilC overexpression strain N560 (Rudel et al., see above)from Neisseria gonorrhoeae had been extracted, the extract was loaded onan Ni-NTA chromatography matrix. Usually, for the method that wasdeveloped for the purification of recombinant PilC unspecifically boundmaterial was removed by extensive washing with a buffer containingimidazole. However, in the first elution fraction a protein of 36 kd(OrfA) could be identified together with PilC in an approximatelyequimolar ratio.

For the preparation of the PilC-OrfA protein fraction the strain N560from Neisseria gonorrhoeae was plated on 30 GC-agar plates and incubatedin 5% CO₂ at 37° C. for 20 hours. The GC-agar medium (GC agar base,Becton Dickinson, Heidelberg) contained the usual additional factorsnecessary for the growth of Neisseria gonorrhoeae (0.1 mg vitamin B12,10 mg adenine, 0.3 mg guanine, 100 mg glutamine, 1 mg cocarboxylase, 0.3mg thiamine, 259 mg L-cysteine, 11 mg L-cystine, 1.5 mg arginine, 5 mguracil, 0.2 mg Fe(NO₃)₃, 2.5 mg nicotineamide-adenine dinucleotide, 0.13mg p-aminobenzoic acid and 1 g dextrose per 1 liter of medium) that wereadded as a sterile filtrate to the GC basis medium after heatsterilization. Furthermore, the so supplemented GC agar medium contained5 μg/ml tetracycline and 100 μM IPTG. The bacterial lawns were removedwith cotton pads, transferred to 30 ml of washing buffer (Tris-HCl pH8.0 with 0.15 M NaCl) and centrifuged at 4,000 rpm, 4° C. for 15 minutes(Du Pont Sorvall Centrifuge RC-5B, Rotor SS-34). The cell sediment wasagain resuspended in 30 ml of washing buffer, and the bacteria werebroken up by ultrasonic homogenization after lysozyme and 5 mM EDTA Na₂had been added. Intact bacteria were separated by centrifugation at5,000 rpm at 4° C. for 15 minutes. The cell coats of the lysed bacteriawere sedimented by centrifugation of the supernatant at 20,000 rpm at 4°C. for 60 minutes and taken up in 10 ml of washing buffer additionallycontaining 10% glycerine, 10 mM MgCl₂ and 2% Triton X-100. After anincubation of 45 minutes at 37° C. they were centrifuged again (20,000rpm, 4° C. for 60 minutes) and the membrane sediment suspended in 10 mlof washing buffer with 10% glycerine, 10 mM MgCl₂ and 2% LDAO(N,N-Dimethyldodecylamin-N-oxide) and incubated at 37° C. for 60minutes. After they were centrifuged again (20,000 rpm, 4° C. for 60minutes), the supernatant containing the biologically active PilC-OrfAcomplex was subjected to a nickel-chelate-affinity chromatography forfurther purification. For this purpose a Ni-NTA-gel matrix (300 ml bedvolume) was washed with 5 bed volumes of aqua bidest. and loaded with 10ml of the supernatant. Unspecifically bound proteins were removed byelution with 5 column volumes of 50 mM imidazole in PBS buffer pH 8.0.After the column had been washed again with 5 to 10 bed volumes 20 mMsodium phosphate pH 7.5 with 0.15 M NaCl (PBS buffer) the biologicallyactive PilC-OrfA complex was eluted with a citrate/phosphate buffer (10mM citric acid, 1 M sodium phosphate, pH 3.5, 10% glycerin, 0.15 M NaCl)in the first elution fraction and instantly neutralized with a 1 MNa₂HPO₄ solution. The eluate containing PilC and OrfA was frozen inliquid nitrogen and strored at −70° C.

Example 2 Isolation of the Polynucleotide Sequence Carrying the Orf-geneRegion

To further characterize the 36 kd OrfA protein, mice were immunized withthe PilC-36 kd protein fraction. The 36 kd protein proved to be veryimmunogenic. With the antibodies obtained this way a pBA plasmid genelibrary of the Neisseria gonorrhoeae MS11 genome in E. coli GC1 wasscreened for the presence of antigens. Several clones showing a positivereaction were isolated and clone H1967 was chosen for furthercharacterization.

The library plasmid pES25 (FIG. 1) of clone H1967 contained a genomicfragment of approximately 11 kb, cloned in vector pBA. Restrictionfragments of the total region were subcloned in pUC and pBluescript KS(+) vectors, respectively. On the basis of the expression of the derivedplasmids in minicells and immunoblotting analyses subclones were chosenproducing the 36 kd protein. The subclones were used for sequencing. Thesequences were determined by directly sequencing restriction fragments,by sequencing continuously shortened ExoIII nuclease fragments of theBgII-PstI fragment (positions 1 to 2560 of SEQ ID NO: 1), as well as bysequencing PCR amplified fragments.

The region depicted in SEQ ID No. 1 starting from the BgII cleavage site(position 1) to the HindIII cleavage site (position 3260) had three openreading frames with a high coding probability with each reading framebeginning with the start codon ATG, having a ribosome binding site thatprecedes the start codon in a suitable distance (S.D. sequence) andending with a stop codon.

The three reading frames have the same orientation. The first openreading frame starts at position 136 of the sequence depicted in SEQ IDNo. 1 and ends at position 450 with the stop codon TAA. The encodedprotein was called OrfI and had an apparent molecular weight of 18 kd inthe T7 expression system.

No significant homologues could be identified by sequence comparison inthe EMBL gene library (Release 43.0 from 6/95) and in the SwissProt databank (Release 31.0 from 3/95), neither on a nucleotide sequence levelnor on an amino acid sequence level.

The second open reading frame starts at position 583 and ends atposition 1545 with the stop codon TGA. It encodes the OrfA proteinhaving an apparent molecular weight of 36 kd in the T7 expressionsystem. Also to this sequence no significant homologues could bedetected via data base search. The sequence analysis by means of theprotein analysis program “Motifs” (GCG Genetics Computer Group, Inc.,Madison, Wis., USA) showed, however, a complete homology of theN-terminus of OrfA to lipoprotein specific signal sequences (position583 to 636). The characterization of OrfA as a lipoprotein could besubstantiated by experiments (vide infra).

The third open reading frame starts at position 1585 and ends atposition 3114 with the stop codon TGA. The protein OrfB hereby encodedhas an apparent molecular weight of 57 kd in the T7 expression system.Also to this reading frame no homologue could be identified via database search.

As a structural peculiarity the amino terminus of the OrfB sequencedisplays a signal sequence showing similarities to the type IV-prepilinsignal sequence. At positions 8 and 12 of the amino acid sequence thereis phenylalanine so that there are in addition two possible cleavagesites for the type IV pilin signal peptidase. It can be derived herefromthat OrfB presumably is a secreted protein.

The molecular weights of all the three gene products measured in the T7expression system correspond to the values theoretically calculated fromthe sequence. The separation of the expression products by means of gelelectrophoresis showed that the OrfB-band was significantly weaker thanthe OrfA-band in all the cases. This points to a weaker expression ofOrfB.

Two regions showing a sequence homology to the promoter regions wereidentified. One of them is located in front of the orfI gene, the secondone in front of the orfA gene, each leaving an appropriate distance (SEQID No. 1). Therefore, it can be assumed that orfA and orfB form atranscription unity.

The analysis of the Neisseria gonorrhoeae MS11 genome after ClaI andMluI digestion showed a complex band pattern in Southern hybridizationwith plasmid pES-8 as sample. This fact indicates the existence ofseveral copies of the orf-gene region, probably of three copies, in thegenome of Neisseria gonorrhoeae MS11. If all these loci are expressed,if they are subjected to antigenic variations like, for example, theNeisseria genes pilS and opa, and if the flanking regions of the orfgene region are involved in the sequence repetitions, is presently notknown.

Example 3 Characterization of the Localization of OrfA and OrfB on theCell Surface

In order to experimentally prove the lipoprotein nature of orfAderivable from the perfect structure homology of the amino terminus oforfA to lipoprotein signal sequences, both N. gonorrhoeae and E. colirecombinants transformed with the orf-gene region were labeled with [³H]palmitate. The results of the labeling show that in all the cases, bothwith N. gonorrhoeae and with the E. coli recombinants, lipoproteins inthe corresponding molecular weight range could be identified. While withN. gonorrhoeae several proteins were labeled and the labeled band couldnot be precisely assigned since there was no orfA⁻ mutant available, theorfA recombinants of E. coli showed in comparison to the control strainunambiguously only one additional band having the molecular weight ofOrfA. An OrfA fusion protein that was tested in addition and wasamplified at the carboxy-terminal by a fusion of 3 kd, also had a [³H]palmitate labeling and migrated to a position precisely corresponding tothe molecular weight that was, as expected, increased due to the fusion.

When prepared cell coats were treated with detergents, OrfA showed asolubility that is typical of proteins of the outer membrane. Byseparating the cell coat by means of density-gradient centrifugation itcould be confirmed by means of marker proteins that OrfA was located inthe outer membrane of N. gonorrhoeae. Also with orf recombinants of E.coli, OrfA was shown to be a protein component of the outer membrane bymeans of said method.

The accessibility of the cell surface was proven by means of animmunofluorescence test both for OrfA and OrfB. A defective pilC mutantof Neisseria gonorrhoeae the two pilC genes of which were switched offis labeled by the PilC-OrfA antiserum in the same way as recombinant E.coli strains carrying the orf-gene region. The non-transformed controlstrain showed, as was to be expected, a negative reaction. A positivereaction in the immunofluorescence test of N. gonorrhoeae and orfrecombinant E. coli strains could be brought about by means of OrfA andOrfB specific antisera using purified fusion proteins of either OrfA orOrfB for the production of these antisera. If antisera were useddirected against an OrfI fusion protein, the immunofluorescence testwith N. gonorrhoeae was negative. From this it can be deduced that OrfAand OrfB are located on the cell surface and are accessible from theoutside, whereas OrfI probably is located intracellularly.

The surface localization of OrfA and OrfB could only be proven inrecombinant E. coli strains carrying the whole orf region.

Example 4 Adhesin Property of the OrfA-PilC Complex

As mentioned above OrfA could be obtained in pure form by chromatographyon an Ni-NTA-chelate matrix due to its affinity to PilC. Since thefunction of PilC as pilus associated adhesin had been proven and thedirect binding of PilC to human ME-180 cells had been known, it wasobvious to test the adherence property of the PilC-OrfA complex. Theexperiments were performed with the E. coli strain HB101 (E141) since itdoes not possess the mannose specific typel pili and shows almost nobinding to human ME-180 and Chang epithelial cells. After thetransformation of HB101 with the plasmid pES25, no adherence, neither toME-180 nor to Chang cells, could be mediated. If the same recombinants,however, were pre-incubated with PilC protein, a strong adherence toChang epithelial cells but not to ME-180 cells could be induced (TableI).

TABLE I OrfA-dependent modulation of the PilC mediated adhesin functionAdherence to human epithelial cells ME180 cells Chang cells N.gonorrhoeae, Orf+ PilC+, Pili+ + + + + N. gonorrhoeae, Orf+, PilC+,Pili− + + + + E. coli (E141) − − E. coli (E141) + PilC (extern) − − E.coli (H2561) − − E. coli (H2561) + PilC (extern) − − E. coli (H2560) − +E. coli (H2560) + PilC (extern) − + + +

Three independent experiments were evaluated, whereby the adherence ofNeisseria was determined using 500 cells and the adherence of the E.coli strains was determined per epithelial cell.

+++100%, ++50%, +25% adherence.

E. coli E141=E. coli strain HB101 without plasmid; E. coli H2561=E. colistrain HB101 with plasmid pBA; E. coli H2560=E. coli strain HB101 withplasmid pES25. The plasmid PES25 (FIG. 1) is a pBA vector containing agenomic fragment from Neisseria gonorrhoeae of approximately 11 kbcarrying the coding regions orfA, orfB and orfI.

The E. coli strain H2560 was deposited at the Deutsche Sammlung fürMikroorganismen (DSM, Braunschweig, Germany) under the DSM-AccessionNumber DSM 10257.

The result obtained is surprising since pilus carrying Neisseria bind toME-180 cells with a significantly higher affinity than to Changepithelial cells. This result can be put down to the fact that PilC hasdifferent adherence properties depending on its localization. As anadhesin component in the pilus PilC preferably binds to receptors of theME-1800 cell surface, whereas as an adhesin located on the cell surfacein the complex with OrfA PilC preferably recognizes receptors on Changepithelial cells. If in the latter case adhesin properties also can beascribed to OrfA and/or OrfB, is presently not known.

The results obtained for recombinant E. coli strains could be reproducedwith the same result with N. gonorrhoea. If the pilus-free strain N 300(P-Opa-), which hardly binds to ME-180 or Chang cells, is pre-incubatedwith purified PilC, the adherence to Chang epithelial cells can besignificantly increased.

The described experimental approaches obviously provide for a model thatis suitable to analyze a mechanism for the modulation of the adherenceproperties, how they can in cascade-like order effect the increasinglystrong adherence of the pathogens to the host cells or how they can bethe basis for the tissue tropism.

11 1 3287 DNA Neisseria gonorrhoeae CDS (136)..(447) CDS (583)..(1542)CDS (1585)..(3111) 1 cggcgcaaac ggcggacgct gctgttagcc ccgcttgaaacaaatgccgt ctgaacgcca 60 cttcagacgg catttttata ataaggcgct gtcctagataactagggaaa ttcaaattaa 120 gttagaatta tccct atg aga aaa agc cgt cta agccgg tat aaa caa aat 171 Met Arg Lys Ser Arg Leu Ser Arg Tyr Lys Gln Asn1 5 10 aaa ctc att gaa ctg ttt gtc gca ggc gta act gca aga aca gca gca219 Lys Leu Ile Glu Leu Phe Val Ala Gly Val Thr Ala Arg Thr Ala Ala 1520 25 gag cct gac agc att gtt tat acg gat tgt tat cgt cgc tat gat gta267 Glu Pro Asp Ser Ile Val Tyr Thr Asp Cys Tyr Arg Arg Tyr Asp Val 3035 40 ttg gat gcg ggc gaa ttt agc cat ttc cgt atc aat cac agc aca cat315 Leu Asp Ala Gly Glu Phe Ser His Phe Arg Ile Asn His Ser Thr His 4550 55 60 ttt gcc gaa cga caa aac cat att aat gga att ggg aac ttt tgg aac363 Phe Ala Glu Arg Gln Asn His Ile Asn Gly Ile Gly Asn Phe Trp Asn 6570 75 cgg gca aaa cgt cat tta cgc aag ttt gac ggc att ccc aaa gag cat411 Arg Ala Lys Arg His Leu Arg Lys Phe Asp Gly Ile Pro Lys Glu His 8085 90 ttt gag ccg tat tta aag gag tgc gaa cgg cgt ttt taacaacagt 457 PheGlu Pro Tyr Leu Lys Glu Cys Glu Arg Arg Phe 95 100 gagataaaag ttcttgttccattttaaaac aattagtaaa atcgagttta tcctagttgt 517 ccaggacggc ccctaatttatttacaattt tgatacaatt tgtttttcat caaaggagaa 577 aatct atg cgg gca cggctg ctg ata cct att ctt ttt tcg gtt ttt att 627 Met Arg Ala Arg Leu LeuIle Pro Ile Leu Phe Ser Val Phe Ile 105 110 115 tta tcc gcc tgc ggg acactg aca ggt att cca tcg cat ggc gga ggc 675 Leu Ser Ala Cys Gly Thr LeuThr Gly Ile Pro Ser His Gly Gly Gly 120 125 130 135 aaa cgc ttc gcg gtcgaa caa gaa ctt gtg gcc gct tct gcc aga gct 723 Lys Arg Phe Ala Val GluGln Glu Leu Val Ala Ala Ser Ala Arg Ala 140 145 150 gcc gtt aaa gac atggat tta cag gca tta cac gga cga aaa gtt gca 771 Ala Val Lys Asp Met AspLeu Gln Ala Leu His Gly Arg Lys Val Ala 155 160 165 ttg tac att gca actatg ggc gac caa ggt tca ggc agt ttg aca ggg 819 Leu Tyr Ile Ala Thr MetGly Asp Gln Gly Ser Gly Ser Leu Thr Gly 170 175 180 ggt cgc tac tcc attgat gca ctg att cgc ggc gaa tac ata aac agc 867 Gly Arg Tyr Ser Ile AspAla Leu Ile Arg Gly Glu Tyr Ile Asn Ser 185 190 195 cct gcc gtc cgc accgat tac acc tat ccg cgt tac gaa acc acc gct 915 Pro Ala Val Arg Thr AspTyr Thr Tyr Pro Arg Tyr Glu Thr Thr Ala 200 205 210 215 gaa aca aca tcaggc ggt ttg acg ggt tta acc act tct tta tct aca 963 Glu Thr Thr Ser GlyGly Leu Thr Gly Leu Thr Thr Ser Leu Ser Thr 220 225 230 ctt aat gcc cctgca ctc tcg cgc acc caa tca gac ggt agc gga agt 1011 Leu Asn Ala Pro AlaLeu Ser Arg Thr Gln Ser Asp Gly Ser Gly Ser 235 240 245 agg agc agt ctgggc tta aat att ggc ggg atg ggg gat tat cga aat 1059 Arg Ser Ser Leu GlyLeu Asn Ile Gly Gly Met Gly Asp Tyr Arg Asn 250 255 260 gaa acc ttg acgacc aac ccg cgc gac act gcc ttt ctt tcc cac ttg 1107 Glu Thr Leu Thr ThrAsn Pro Arg Asp Thr Ala Phe Leu Ser His Leu 265 270 275 gta cag acc gtattt ttc ctg cgc ggc ata gac gtt gtt tct cct gcc 1155 Val Gln Thr Val PhePhe Leu Arg Gly Ile Asp Val Val Ser Pro Ala 280 285 290 295 aat gcc gataca gat gtg ttt att aac atc gac gta ttc gga acg ata 1203 Asn Ala Asp ThrAsp Val Phe Ile Asn Ile Asp Val Phe Gly Thr Ile 300 305 310 cgc aac agaacc gaa atg cac cta tac aat gcc gaa aca ctg aaa gcc 1251 Arg Asn Arg ThrGlu Met His Leu Tyr Asn Ala Glu Thr Leu Lys Ala 315 320 325 caa aca aaactg gaa tat ttc gca gta gac aga acc aat aaa aaa ttg 1299 Gln Thr Lys LeuGlu Tyr Phe Ala Val Asp Arg Thr Asn Lys Lys Leu 330 335 340 ctc atc aaaccc aaa acc aat gcg ttt gaa gct gcc tat aaa gaa aat 1347 Leu Ile Lys ProLys Thr Asn Ala Phe Glu Ala Ala Tyr Lys Glu Asn 345 350 355 tac gca ttgtgg atg ggg ccg tat aaa gta agc aaa gga atc aaa ccg 1395 Tyr Ala Leu TrpMet Gly Pro Tyr Lys Val Ser Lys Gly Ile Lys Pro 360 365 370 375 acg gaagga tta atg gtc gat ttc tcc gat atc cgg cca tac ggc aat 1443 Thr Glu GlyLeu Met Val Asp Phe Ser Asp Ile Arg Pro Tyr Gly Asn 380 385 390 cat acgggt aac tcc gcc cca tcc gta gag gct gat aac agt cat gag 1491 His Thr GlyAsn Ser Ala Pro Ser Val Glu Ala Asp Asn Ser His Glu 395 400 405 ggg tatgga tac agc gat gaa gca gtg cga caa cat aga caa ggg caa 1539 Gly Tyr GlyTyr Ser Asp Glu Ala Val Arg Gln His Arg Gln Gly Gln 410 415 420 ccttgattcacac tgccataacc gcttgctgcc aaggaaaaca aa atg aat ttg 1593 Pro MetAsn Leu 425 cct att caa aaa ttc atg atg ctg ttt gca gcg gca ata tcg ttgctg 1641 Pro Ile Gln Lys Phe Met Met Leu Phe Ala Ala Ala Ile Ser Leu Leu430 435 440 caa atc ccc att agt cat gcg aac ggt ttg gat gcc cgt ttg cgcgat 1689 Gln Ile Pro Ile Ser His Ala Asn Gly Leu Asp Ala Arg Leu Arg Asp445 450 455 gat atg cag gca aaa cac tac gaa ccg ggt ggc aaa tac cat ctgttc 1737 Asp Met Gln Ala Lys His Tyr Glu Pro Gly Gly Lys Tyr His Leu Phe460 465 470 475 ggt aat gct cgc ggc agt gtt aaa aat cgg gtt tgc gcc gtccaa aca 1785 Gly Asn Ala Arg Gly Ser Val Lys Asn Arg Val Cys Ala Val GlnThr 480 485 490 ttt gat gca act gcg gtc ggc ccc ata ctg cct att aca cacgaa cgg 1833 Phe Asp Ala Thr Ala Val Gly Pro Ile Leu Pro Ile Thr His GluArg 495 500 505 aca ggg ttt gaa ggc att atc ggt tat gaa acc cat ttt tcagga cac 1881 Thr Gly Phe Glu Gly Ile Ile Gly Tyr Glu Thr His Phe Ser GlyHis 510 515 520 gga cac gaa gta cac agt ccg ttc gat aat cat gat tca aaaagc act 1929 Gly His Glu Val His Ser Pro Phe Asp Asn His Asp Ser Lys SerThr 525 530 535 tct gat ttc agc ggc ggc gta gac ggc ggt ttt acc gtt taccaa ctt 1977 Ser Asp Phe Ser Gly Gly Val Asp Gly Gly Phe Thr Val Tyr GlnLeu 540 545 550 555 cat cgg aca ggg tcg gaa ata cat ccc gca gac gga tatgac ggg cct 2025 His Arg Thr Gly Ser Glu Ile His Pro Ala Asp Gly Tyr AspGly Pro 560 565 570 caa ggc ggc ggt tat ccg gaa cca caa ggg gca agg gatata tac agc 2073 Gln Gly Gly Gly Tyr Pro Glu Pro Gln Gly Ala Arg Asp IleTyr Ser 575 580 585 tac cat atc aaa gga act tca acc aaa aca aag ata aacact gtt ccg 2121 Tyr His Ile Lys Gly Thr Ser Thr Lys Thr Lys Ile Asn ThrVal Pro 590 595 600 caa gcc cct ttt tca gac cgc tgg cta aaa gaa aat gccggt gcc gct 2169 Gln Ala Pro Phe Ser Asp Arg Trp Leu Lys Glu Asn Ala GlyAla Ala 605 610 615 tcc ggt ttt ctc agc cgt gcg gat gaa gca gga aaa ctgata tgg gaa 2217 Ser Gly Phe Leu Ser Arg Ala Asp Glu Ala Gly Lys Leu IleTrp Glu 620 625 630 635 aac gac ccc gat aaa aat tgg cgg gct aac cgt atggat gat att cgc 2265 Asn Asp Pro Asp Lys Asn Trp Arg Ala Asn Arg Met AspAsp Ile Arg 640 645 650 ggc atc gtc caa ggt gcg gtt aat cct ttt tta acgggt ttt cag gga 2313 Gly Ile Val Gln Gly Ala Val Asn Pro Phe Leu Thr GlyPhe Gln Gly 655 660 665 ttg gga gtt ggg gca att aca gac agt gcg gta agcccg gta acc tat 2361 Leu Gly Val Gly Ala Ile Thr Asp Ser Ala Val Ser ProVal Thr Tyr 670 675 680 gcg gca gca cgg aaa act tta cag ggt att cac aattta gga aat tta 2409 Ala Ala Ala Arg Lys Thr Leu Gln Gly Ile His Asn LeuGly Asn Leu 685 690 695 agt ccg gaa gca caa ctt gcc gcc gcg agc cta ttacag gac agt gcc 2457 Ser Pro Glu Ala Gln Leu Ala Ala Ala Ser Leu Leu GlnAsp Ser Ala 700 705 710 715 ttt gcg gta aaa gac ggc atc aat tcc gcc agacaa tgg gct gat gcc 2505 Phe Ala Val Lys Asp Gly Ile Asn Ser Ala Arg GlnTrp Ala Asp Ala 720 725 730 cat ccg aat ata aca gca aca gcc caa act gccctt gcc gta gca gag 2553 His Pro Asn Ile Thr Ala Thr Ala Gln Thr Ala LeuAla Val Ala Glu 735 740 745 gct gca ggt acg gtt tgg gga ggt aaa aaa gtagaa ctt aac ccg acc 2601 Ala Ala Gly Thr Val Trp Gly Gly Lys Lys Val GluLeu Asn Pro Thr 750 755 760 aaa tgg gat tgg gtt aaa aat acc ggc tat gaaaaa cct gct gcc cga 2649 Lys Trp Asp Trp Val Lys Asn Thr Gly Tyr Glu LysPro Ala Ala Arg 765 770 775 cct atg cag act gta gac ggg gaa atg gcc gggaaa aat aag cca ccg 2697 Pro Met Gln Thr Val Asp Gly Glu Met Ala Gly LysAsn Lys Pro Pro 780 785 790 795 aaa cca agt acg cag caa cac tct aca cactct gat aac aat atc ggc 2745 Lys Pro Ser Thr Gln Gln His Ser Thr His SerAsp Asn Asn Ile Gly 800 805 810 tta cct gcc cca tat gtt aaa cct gat acatct att tct ccg aca gga 2793 Leu Pro Ala Pro Tyr Val Lys Pro Asp Thr SerIle Ser Pro Thr Gly 815 820 825 aca att caa gac cgc atc aga tgg aca aaatcc aag ttt cct act gag 2841 Thr Ile Gln Asp Arg Ile Arg Trp Thr Lys SerLys Phe Pro Thr Glu 830 835 840 aaa tct tta aat gga cat ttc aaa gct catgga aaa gaa ttt ggc gat 2889 Lys Ser Leu Asn Gly His Phe Lys Ala His GlyLys Glu Phe Gly Asp 845 850 855 ata acc att gaa gac tac caa aaa atg gcgtct gat ttg tta tca aaa 2937 Ile Thr Ile Glu Asp Tyr Gln Lys Met Ala SerAsp Leu Leu Ser Lys 860 865 870 875 cag aca tcg gac aag ata tta ggt tatcag acg gaa cat aga cga gtg 2985 Gln Thr Ser Asp Lys Ile Leu Gly Tyr GlnThr Glu His Arg Arg Val 880 885 890 cgc tat gat atc aat aac aat atc tatgtt ttg gcc aat cca aaa aca 3033 Arg Tyr Asp Ile Asn Asn Asn Ile Tyr ValLeu Ala Asn Pro Lys Thr 895 900 905 ttc aaa atc aaa aca atg ttt aaa ccaaac tta gga aag gag tat tat 3081 Phe Lys Ile Lys Thr Met Phe Lys Pro AsnLeu Gly Lys Glu Tyr Tyr 910 915 920 gat gga gaa ttc aaa aaa gac atg ggaaat tgacggagaa atatggctac 3131 Asp Gly Glu Phe Lys Lys Asp Met Gly Asn925 930 attgtcctgt ttgcggaact gaagttatgg actatgatat ctgtgacgtttgtcagtggc 3191 aaaatacagg agaaactaat atagatggtg gtcctaatga aatgacacttgcggaggcga 3251 aagaagctta cgcaaaaggc ttaccaatca gataaa 3287 2 2 ThisSequence is intentionally skipped 3 104 PRT Neisseria gonorrhoeae 3 MetArg Lys Ser Arg Leu Ser Arg Tyr Lys Gln Asn Lys Leu Ile Glu 1 5 10 15Leu Phe Val Ala Gly Val Thr Ala Arg Thr Ala Ala Glu Pro Asp Ser 20 25 30Ile Val Tyr Thr Asp Cys Tyr Arg Arg Tyr Asp Val Leu Asp Ala Gly 35 40 45Glu Phe Ser His Phe Arg Ile Asn His Ser Thr His Phe Ala Glu Arg 50 55 60Gln Asn His Ile Asn Gly Ile Gly Asn Phe Trp Asn Arg Ala Lys Arg 65 70 7580 His Leu Arg Lys Phe Asp Gly Ile Pro Lys Glu His Phe Glu Pro Tyr 85 9095 Leu Lys Glu Cys Glu Arg Arg Phe 100 4 320 PRT Neisseria gonorrhoeae 4Met Arg Ala Arg Leu Leu Ile Pro Ile Leu Phe Ser Val Phe Ile Leu 1 5 1015 Ser Ala Cys Gly Thr Leu Thr Gly Ile Pro Ser His Gly Gly Gly Lys 20 2530 Arg Phe Ala Val Glu Gln Glu Leu Val Ala Ala Ser Ala Arg Ala Ala 35 4045 Val Lys Asp Met Asp Leu Gln Ala Leu His Gly Arg Lys Val Ala Leu 50 5560 Tyr Ile Ala Thr Met Gly Asp Gln Gly Ser Gly Ser Leu Thr Gly Gly 65 7075 80 Arg Tyr Ser Ile Asp Ala Leu Ile Arg Gly Glu Tyr Ile Asn Ser Pro 8590 95 Ala Val Arg Thr Asp Tyr Thr Tyr Pro Arg Tyr Glu Thr Thr Ala Glu100 105 110 Thr Thr Ser Gly Gly Leu Thr Gly Leu Thr Thr Ser Leu Ser ThrLeu 115 120 125 Asn Ala Pro Ala Leu Ser Arg Thr Gln Ser Asp Gly Ser GlySer Arg 130 135 140 Ser Ser Leu Gly Leu Asn Ile Gly Gly Met Gly Asp TyrArg Asn Glu 145 150 155 160 Thr Leu Thr Thr Asn Pro Arg Asp Thr Ala PheLeu Ser His Leu Val 165 170 175 Gln Thr Val Phe Phe Leu Arg Gly Ile AspVal Val Ser Pro Ala Asn 180 185 190 Ala Asp Thr Asp Val Phe Ile Asn IleAsp Val Phe Gly Thr Ile Arg 195 200 205 Asn Arg Thr Glu Met His Leu TyrAsn Ala Glu Thr Leu Lys Ala Gln 210 215 220 Thr Lys Leu Glu Tyr Phe AlaVal Asp Arg Thr Asn Lys Lys Leu Leu 225 230 235 240 Ile Lys Pro Lys ThrAsn Ala Phe Glu Ala Ala Tyr Lys Glu Asn Tyr 245 250 255 Ala Leu Trp MetGly Pro Tyr Lys Val Ser Lys Gly Ile Lys Pro Thr 260 265 270 Glu Gly LeuMet Val Asp Phe Ser Asp Ile Arg Pro Tyr Gly Asn His 275 280 285 Thr GlyAsn Ser Ala Pro Ser Val Glu Ala Asp Asn Ser His Glu Gly 290 295 300 TyrGly Tyr Ser Asp Glu Ala Val Arg Gln His Arg Gln Gly Gln Pro 305 310 315320 5 509 PRT Neisseria gonorrhoeae 5 Met Asn Leu Pro Ile Gln Lys PheMet Met Leu Phe Ala Ala Ala Ile 1 5 10 15 Ser Leu Leu Gln Ile Pro IleSer His Ala Asn Gly Leu Asp Ala Arg 20 25 30 Leu Arg Asp Asp Met Gln AlaLys His Tyr Glu Pro Gly Gly Lys Tyr 35 40 45 His Leu Phe Gly Asn Ala ArgGly Ser Val Lys Asn Arg Val Cys Ala 50 55 60 Val Gln Thr Phe Asp Ala ThrAla Val Gly Pro Ile Leu Pro Ile Thr 65 70 75 80 His Glu Arg Thr Gly PheGlu Gly Ile Ile Gly Tyr Glu Thr His Phe 85 90 95 Ser Gly His Gly His GluVal His Ser Pro Phe Asp Asn His Asp Ser 100 105 110 Lys Ser Thr Ser AspPhe Ser Gly Gly Val Asp Gly Gly Phe Thr Val 115 120 125 Tyr Gln Leu HisArg Thr Gly Ser Glu Ile His Pro Ala Asp Gly Tyr 130 135 140 Asp Gly ProGln Gly Gly Gly Tyr Pro Glu Pro Gln Gly Ala Arg Asp 145 150 155 160 IleTyr Ser Tyr His Ile Lys Gly Thr Ser Thr Lys Thr Lys Ile Asn 165 170 175Thr Val Pro Gln Ala Pro Phe Ser Asp Arg Trp Leu Lys Glu Asn Ala 180 185190 Gly Ala Ala Ser Gly Phe Leu Ser Arg Ala Asp Glu Ala Gly Lys Leu 195200 205 Ile Trp Glu Asn Asp Pro Asp Lys Asn Trp Arg Ala Asn Arg Met Asp210 215 220 Asp Ile Arg Gly Ile Val Gln Gly Ala Val Asn Pro Phe Leu ThrGly 225 230 235 240 Phe Gln Gly Leu Gly Val Gly Ala Ile Thr Asp Ser AlaVal Ser Pro 245 250 255 Val Thr Tyr Ala Ala Ala Arg Lys Thr Leu Gln GlyIle His Asn Leu 260 265 270 Gly Asn Leu Ser Pro Glu Ala Gln Leu Ala AlaAla Ser Leu Leu Gln 275 280 285 Asp Ser Ala Phe Ala Val Lys Asp Gly IleAsn Ser Ala Arg Gln Trp 290 295 300 Ala Asp Ala His Pro Asn Ile Thr AlaThr Ala Gln Thr Ala Leu Ala 305 310 315 320 Val Ala Glu Ala Ala Gly ThrVal Trp Gly Gly Lys Lys Val Glu Leu 325 330 335 Asn Pro Thr Lys Trp AspTrp Val Lys Asn Thr Gly Tyr Glu Lys Pro 340 345 350 Ala Ala Arg Pro MetGln Thr Val Asp Gly Glu Met Ala Gly Lys Asn 355 360 365 Lys Pro Pro LysPro Ser Thr Gln Gln His Ser Thr His Ser Asp Asn 370 375 380 Asn Ile GlyLeu Pro Ala Pro Tyr Val Lys Pro Asp Thr Ser Ile Ser 385 390 395 400 ProThr Gly Thr Ile Gln Asp Arg Ile Arg Trp Thr Lys Ser Lys Phe 405 410 415Pro Thr Glu Lys Ser Leu Asn Gly His Phe Lys Ala His Gly Lys Glu 420 425430 Phe Gly Asp Ile Thr Ile Glu Asp Tyr Gln Lys Met Ala Ser Asp Leu 435440 445 Leu Ser Lys Gln Thr Ser Asp Lys Ile Leu Gly Tyr Gln Thr Glu His450 455 460 Arg Arg Val Arg Tyr Asp Ile Asn Asn Asn Ile Tyr Val Leu AlaAsn 465 470 475 480 Pro Lys Thr Phe Lys Ile Lys Thr Met Phe Lys Pro AsnLeu Gly Lys 485 490 495 Glu Tyr Tyr Asp Gly Glu Phe Lys Lys Asp Met GlyAsn 500 505 6 1136 DNA Neisseria gonorrhoeae CDS (135)..(1094) 6aacaacagtg agataaaagt tcttgttcca ttttaaaaca attagtaaaa tcgagtttat 60cctagttgtc caggacggcc cctaatttat ttacaatttt gatacaattt gtttttcatc 120aaaggagaaa atct atg cgg gca cgg ctg ctg ata cct att ctt ttt tcg 170 MetArg Ala Arg Leu Leu Ile Pro Ile Leu Phe Ser 1 5 10 gtt ttt att tta tccgcc tgc ggg aca ctg aca ggt att cca tcg cat 218 Val Phe Ile Leu Ser AlaCys Gly Thr Leu Thr Gly Ile Pro Ser His 15 20 25 ggc gga ggc aaa cgc ttcgcg gtc gaa caa gaa ctt gtg gcc gct tct 266 Gly Gly Gly Lys Arg Phe AlaVal Glu Gln Glu Leu Val Ala Ala Ser 30 35 40 gcc aga gct gcc gtt aaa gacatg gat tta cag gca tta cac gga cga 314 Ala Arg Ala Ala Val Lys Asp MetAsp Leu Gln Ala Leu His Gly Arg 45 50 55 60 aaa gtt gca ttg tac att gcaact atg ggc gac caa ggt tca ggc agt 362 Lys Val Ala Leu Tyr Ile Ala ThrMet Gly Asp Gln Gly Ser Gly Ser 65 70 75 ttg aca ggg ggt cgc tac tcc attgat gca ctg att cgc ggc gaa tac 410 Leu Thr Gly Gly Arg Tyr Ser Ile AspAla Leu Ile Arg Gly Glu Tyr 80 85 90 ata aac agc cct gcc gtc cgc acc gattac acc tat ccg cgt tac gaa 458 Ile Asn Ser Pro Ala Val Arg Thr Asp TyrThr Tyr Pro Arg Tyr Glu 95 100 105 acc acc gct gaa aca aca tca ggc ggtttg acg ggt tta acc act tct 506 Thr Thr Ala Glu Thr Thr Ser Gly Gly LeuThr Gly Leu Thr Thr Ser 110 115 120 tta tct aca ctt aat gcc cct gca ctctcg cgc acc caa tca gac ggt 554 Leu Ser Thr Leu Asn Ala Pro Ala Leu SerArg Thr Gln Ser Asp Gly 125 130 135 140 agc gga agt agg agc agt ctg ggctta aat att ggc ggg atg ggg gat 602 Ser Gly Ser Arg Ser Ser Leu Gly LeuAsn Ile Gly Gly Met Gly Asp 145 150 155 tat cga aat gaa acc ttg acg accaac ccg cgc gac act gcc ttt ctt 650 Tyr Arg Asn Glu Thr Leu Thr Thr AsnPro Arg Asp Thr Ala Phe Leu 160 165 170 tcc cac ttg gta cag acc gta tttttc ctg cgc ggc ata gac gtt gtt 698 Ser His Leu Val Gln Thr Val Phe PheLeu Arg Gly Ile Asp Val Val 175 180 185 tct cct gcc aat gcc gat aca gatgtg ttt att aac atc gac gta ttc 746 Ser Pro Ala Asn Ala Asp Thr Asp ValPhe Ile Asn Ile Asp Val Phe 190 195 200 gga acg ata cgc aac aga acc gaaatg cac cta tac aat gcc gaa aca 794 Gly Thr Ile Arg Asn Arg Thr Glu MetHis Leu Tyr Asn Ala Glu Thr 205 210 215 220 ctg aaa gcc caa aca aaa ctggaa tat ttc gca gta gac aga acc aat 842 Leu Lys Ala Gln Thr Lys Leu GluTyr Phe Ala Val Asp Arg Thr Asn 225 230 235 aaa aaa ttg ctc atc aaa cccaaa acc aat gcg ttt gaa gct gcc tat 890 Lys Lys Leu Leu Ile Lys Pro LysThr Asn Ala Phe Glu Ala Ala Tyr 240 245 250 aaa gaa aat tac gca ttg tggatg ggg ccg tat aaa gta agc aaa gga 938 Lys Glu Asn Tyr Ala Leu Trp MetGly Pro Tyr Lys Val Ser Lys Gly 255 260 265 atc aaa ccg acg gaa gga ttaatg gtc gat ttc tcc gat atc cgg cca 986 Ile Lys Pro Thr Glu Gly Leu MetVal Asp Phe Ser Asp Ile Arg Pro 270 275 280 tac ggc aat cat acg ggt aactcc gcc cca tcc gta gag gct gat aac 1034 Tyr Gly Asn His Thr Gly Asn SerAla Pro Ser Val Glu Ala Asp Asn 285 290 295 300 agt cat gag ggg tat ggatac agc gat gaa gca gtg cga caa cat aga 1082 Ser His Glu Gly Tyr Gly TyrSer Asp Glu Ala Val Arg Gln His Arg 305 310 315 caa ggg caa ccttgattcacac tgccataacc gcttgctgcc aaggaaaaca aa 1136 Gln Gly Gln Pro 3207 320 PRT Neisseria gonorrhoeae 7 Met Arg Ala Arg Leu Leu Ile Pro IleLeu Phe Ser Val Phe Ile Leu 1 5 10 15 Ser Ala Cys Gly Thr Leu Thr GlyIle Pro Ser His Gly Gly Gly Lys 20 25 30 Arg Phe Ala Val Glu Gln Glu LeuVal Ala Ala Ser Ala Arg Ala Ala 35 40 45 Val Lys Asp Met Asp Leu Gln AlaLeu His Gly Arg Lys Val Ala Leu 50 55 60 Tyr Ile Ala Thr Met Gly Asp GlnGly Ser Gly Ser Leu Thr Gly Gly 65 70 75 80 Arg Tyr Ser Ile Asp Ala LeuIle Arg Gly Glu Tyr Ile Asn Ser Pro 85 90 95 Ala Val Arg Thr Asp Tyr ThrTyr Pro Arg Tyr Glu Thr Thr Ala Glu 100 105 110 Thr Thr Ser Gly Gly LeuThr Gly Leu Thr Thr Ser Leu Ser Thr Leu 115 120 125 Asn Ala Pro Ala LeuSer Arg Thr Gln Ser Asp Gly Ser Gly Ser Arg 130 135 140 Ser Ser Leu GlyLeu Asn Ile Gly Gly Met Gly Asp Tyr Arg Asn Glu 145 150 155 160 Thr LeuThr Thr Asn Pro Arg Asp Thr Ala Phe Leu Ser His Leu Val 165 170 175 GlnThr Val Phe Phe Leu Arg Gly Ile Asp Val Val Ser Pro Ala Asn 180 185 190Ala Asp Thr Asp Val Phe Ile Asn Ile Asp Val Phe Gly Thr Ile Arg 195 200205 Asn Arg Thr Glu Met His Leu Tyr Asn Ala Glu Thr Leu Lys Ala Gln 210215 220 Thr Lys Leu Glu Tyr Phe Ala Val Asp Arg Thr Asn Lys Lys Leu Leu225 230 235 240 Ile Lys Pro Lys Thr Asn Ala Phe Glu Ala Ala Tyr Lys GluAsn Tyr 245 250 255 Ala Leu Trp Met Gly Pro Tyr Lys Val Ser Lys Gly IleLys Pro Thr 260 265 270 Glu Gly Leu Met Val Asp Phe Ser Asp Ile Arg ProTyr Gly Asn His 275 280 285 Thr Gly Asn Ser Ala Pro Ser Val Glu Ala AspAsn Ser His Glu Gly 290 295 300 Tyr Gly Tyr Ser Asp Glu Ala Val Arg GlnHis Arg Gln Gly Gln Pro 305 310 315 320 8 582 DNA Neisseria gonorrhoeaeCDS (136)..(447) 8 cggcgcaaac ggcggacgct gctgttagcc ccgcttgaaacaaatgccgt ctgaacgcca 60 cttcagacgg catttttata ataaggcgct gtcctagataactagggaaa ttcaaattaa 120 gttagaatta tccct atg aga aaa agc cgt cta agccgg tat aaa caa aat 171 Met Arg Lys Ser Arg Leu Ser Arg Tyr Lys Gln Asn1 5 10 aaa ctc att gaa ctg ttt gtc gca ggc gta act gca aga aca gca gca219 Lys Leu Ile Glu Leu Phe Val Ala Gly Val Thr Ala Arg Thr Ala Ala 1520 25 gag cct gac agc att gtt tat acg gat tgt tat cgt cgc tat gat gta267 Glu Pro Asp Ser Ile Val Tyr Thr Asp Cys Tyr Arg Arg Tyr Asp Val 3035 40 ttg gat gcg ggc gaa ttt agc cat ttc cgt atc aat cac agc aca cat315 Leu Asp Ala Gly Glu Phe Ser His Phe Arg Ile Asn His Ser Thr His 4550 55 60 ttt gcc gaa cga caa aac cat att aat gga att ggg aac ttt tgg aac363 Phe Ala Glu Arg Gln Asn His Ile Asn Gly Ile Gly Asn Phe Trp Asn 6570 75 cgg gca aaa cgt cat tta cgc aag ttt gac ggc att ccc aaa gag cat411 Arg Ala Lys Arg His Leu Arg Lys Phe Asp Gly Ile Pro Lys Glu His 8085 90 ttt gag ccg tat tta aag gag tgc gaa cgg cgt ttt taacaacagt 457 PheGlu Pro Tyr Leu Lys Glu Cys Glu Arg Arg Phe 95 100 gagataaaag ttcttgttccattttaaaac aattagtaaa atcgagttta tcctagttgt 517 ccaggacggc ccctaatttatttacaattt tgatacaatt tgtttttcat caaaggagaa 577 aatct 582 9 104 PRTNeisseria gonorrhoeae 9 Met Arg Lys Ser Arg Leu Ser Arg Tyr Lys Gln AsnLys Leu Ile Glu 1 5 10 15 Leu Phe Val Ala Gly Val Thr Ala Arg Thr AlaAla Glu Pro Asp Ser 20 25 30 Ile Val Tyr Thr Asp Cys Tyr Arg Arg Tyr AspVal Leu Asp Ala Gly 35 40 45 Glu Phe Ser His Phe Arg Ile Asn His Ser ThrHis Phe Ala Glu Arg 50 55 60 Gln Asn His Ile Asn Gly Ile Gly Asn Phe TrpAsn Arg Ala Lys Arg 65 70 75 80 His Leu Arg Lys Phe Asp Gly Ile Pro LysGlu His Phe Glu Pro Tyr 85 90 95 Leu Lys Glu Cys Glu Arg Arg Phe 100 101744 DNA Neisseria gonorrhoeae CDS (42)..(1568) 10 gattcacact gccataaccgcttgctgcca aggaaaacaa a atg aat ttg cct att 56 Met Asn Leu Pro Ile 1 5caa aaa ttc atg atg ctg ttt gca gcg gca ata tcg ttg ctg caa atc 104 GlnLys Phe Met Met Leu Phe Ala Ala Ala Ile Ser Leu Leu Gln Ile 10 15 20 cccatt agt cat gcg aac ggt ttg gat gcc cgt ttg cgc gat gat atg 152 Pro IleSer His Ala Asn Gly Leu Asp Ala Arg Leu Arg Asp Asp Met 25 30 35 cag gcaaaa cac tac gaa ccg ggt ggc aaa tac cat ctg ttc ggt aat 200 Gln Ala LysHis Tyr Glu Pro Gly Gly Lys Tyr His Leu Phe Gly Asn 40 45 50 gct cgc ggcagt gtt aaa aat cgg gtt tgc gcc gtc caa aca ttt gat 248 Ala Arg Gly SerVal Lys Asn Arg Val Cys Ala Val Gln Thr Phe Asp 55 60 65 gca act gcg gtcggc ccc ata ctg cct att aca cac gaa cgg aca ggg 296 Ala Thr Ala Val GlyPro Ile Leu Pro Ile Thr His Glu Arg Thr Gly 70 75 80 85 ttt gaa ggc attatc ggt tat gaa acc cat ttt tca gga cac gga cac 344 Phe Glu Gly Ile IleGly Tyr Glu Thr His Phe Ser Gly His Gly His 90 95 100 gaa gta cac agtccg ttc gat aat cat gat tca aaa agc act tct gat 392 Glu Val His Ser ProPhe Asp Asn His Asp Ser Lys Ser Thr Ser Asp 105 110 115 ttc agc ggc ggcgta gac ggc ggt ttt acc gtt tac caa ctt cat cgg 440 Phe Ser Gly Gly ValAsp Gly Gly Phe Thr Val Tyr Gln Leu His Arg 120 125 130 aca ggg tcg gaaata cat ccc gca gac gga tat gac ggg cct caa ggc 488 Thr Gly Ser Glu IleHis Pro Ala Asp Gly Tyr Asp Gly Pro Gln Gly 135 140 145 ggc ggt tat ccggaa cca caa ggg gca agg gat ata tac agc tac cat 536 Gly Gly Tyr Pro GluPro Gln Gly Ala Arg Asp Ile Tyr Ser Tyr His 150 155 160 165 atc aaa ggaact tca acc aaa aca aag ata aac act gtt ccg caa gcc 584 Ile Lys Gly ThrSer Thr Lys Thr Lys Ile Asn Thr Val Pro Gln Ala 170 175 180 cct ttt tcagac cgc tgg cta aaa gaa aat gcc ggt gcc gct tcc ggt 632 Pro Phe Ser AspArg Trp Leu Lys Glu Asn Ala Gly Ala Ala Ser Gly 185 190 195 ttt ctc agccgt gcg gat gaa gca gga aaa ctg ata tgg gaa aac gac 680 Phe Leu Ser ArgAla Asp Glu Ala Gly Lys Leu Ile Trp Glu Asn Asp 200 205 210 ccc gat aaaaat tgg cgg gct aac cgt atg gat gat att cgc ggc atc 728 Pro Asp Lys AsnTrp Arg Ala Asn Arg Met Asp Asp Ile Arg Gly Ile 215 220 225 gtc caa ggtgcg gtt aat cct ttt tta acg ggt ttt cag gga ttg gga 776 Val Gln Gly AlaVal Asn Pro Phe Leu Thr Gly Phe Gln Gly Leu Gly 230 235 240 245 gtt ggggca att aca gac agt gcg gta agc ccg gta acc tat gcg gca 824 Val Gly AlaIle Thr Asp Ser Ala Val Ser Pro Val Thr Tyr Ala Ala 250 255 260 gca cggaaa act tta cag ggt att cac aat tta gga aat tta agt ccg 872 Ala Arg LysThr Leu Gln Gly Ile His Asn Leu Gly Asn Leu Ser Pro 265 270 275 gaa gcacaa ctt gcc gcc gcg agc cta tta cag gac agt gcc ttt gcg 920 Glu Ala GlnLeu Ala Ala Ala Ser Leu Leu Gln Asp Ser Ala Phe Ala 280 285 290 gta aaagac ggc atc aat tcc gcc aga caa tgg gct gat gcc cat ccg 968 Val Lys AspGly Ile Asn Ser Ala Arg Gln Trp Ala Asp Ala His Pro 295 300 305 aat ataaca gca aca gcc caa act gcc ctt gcc gta gca gag gct gca 1016 Asn Ile ThrAla Thr Ala Gln Thr Ala Leu Ala Val Ala Glu Ala Ala 310 315 320 325 ggtacg gtt tgg gga ggt aaa aaa gta gaa ctt aac ccg acc aaa tgg 1064 Gly ThrVal Trp Gly Gly Lys Lys Val Glu Leu Asn Pro Thr Lys Trp 330 335 340 gattgg gtt aaa aat acc ggc tat gaa aaa cct gct gcc cga cct atg 1112 Asp TrpVal Lys Asn Thr Gly Tyr Glu Lys Pro Ala Ala Arg Pro Met 345 350 355 cagact gta gac ggg gaa atg gcc ggg aaa aat aag cca ccg aaa cca 1160 Gln ThrVal Asp Gly Glu Met Ala Gly Lys Asn Lys Pro Pro Lys Pro 360 365 370 agtacg cag caa cac tct aca cac tct gat aac aat atc ggc tta cct 1208 Ser ThrGln Gln His Ser Thr His Ser Asp Asn Asn Ile Gly Leu Pro 375 380 385 gcccca tat gtt aaa cct gat aca tct att tct ccg aca gga aca att 1256 Ala ProTyr Val Lys Pro Asp Thr Ser Ile Ser Pro Thr Gly Thr Ile 390 395 400 405caa gac cgc atc aga tgg aca aaa tcc aag ttt cct act gag aaa tct 1304 GlnAsp Arg Ile Arg Trp Thr Lys Ser Lys Phe Pro Thr Glu Lys Ser 410 415 420tta aat gga cat ttc aaa gct cat gga aaa gaa ttt ggc gat ata acc 1352 LeuAsn Gly His Phe Lys Ala His Gly Lys Glu Phe Gly Asp Ile Thr 425 430 435att gaa gac tac caa aaa atg gcg tct gat ttg tta tca aaa cag aca 1400 IleGlu Asp Tyr Gln Lys Met Ala Ser Asp Leu Leu Ser Lys Gln Thr 440 445 450tcg gac aag ata tta ggt tat cag acg gaa cat aga cga gtg cgc tat 1448 SerAsp Lys Ile Leu Gly Tyr Gln Thr Glu His Arg Arg Val Arg Tyr 455 460 465gat atc aat aac aat atc tat gtt ttg gcc aat cca aaa aca ttc aaa 1496 AspIle Asn Asn Asn Ile Tyr Val Leu Ala Asn Pro Lys Thr Phe Lys 470 475 480485 atc aaa aca atg ttt aaa cca aac tta gga aag gag tat tat gat gga 1544Ile Lys Thr Met Phe Lys Pro Asn Leu Gly Lys Glu Tyr Tyr Asp Gly 490 495500 gaa ttc aaa aaa gac atg gga aat tgacggagaa atatggctac attgtcctgt1598 Glu Phe Lys Lys Asp Met Gly Asn 505 ttgcggaact gaagttatggactatgatat ctgtgacgtt tgtcagtggc aaaatacagg 1658 agaaactaat atagatggtggtcctaatga aatgacactt gcggaggcga aagaagctta 1718 cgcaaaaggc ttaccaatcagataaa 1744 11 509 PRT Neisseria gonorrhoeae 11 Met Asn Leu Pro Ile GlnLys Phe Met Met Leu Phe Ala Ala Ala Ile 1 5 10 15 Ser Leu Leu Gln IlePro Ile Ser His Ala Asn Gly Leu Asp Ala Arg 20 25 30 Leu Arg Asp Asp MetGln Ala Lys His Tyr Glu Pro Gly Gly Lys Tyr 35 40 45 His Leu Phe Gly AsnAla Arg Gly Ser Val Lys Asn Arg Val Cys Ala 50 55 60 Val Gln Thr Phe AspAla Thr Ala Val Gly Pro Ile Leu Pro Ile Thr 65 70 75 80 His Glu Arg ThrGly Phe Glu Gly Ile Ile Gly Tyr Glu Thr His Phe 85 90 95 Ser Gly His GlyHis Glu Val His Ser Pro Phe Asp Asn His Asp Ser 100 105 110 Lys Ser ThrSer Asp Phe Ser Gly Gly Val Asp Gly Gly Phe Thr Val 115 120 125 Tyr GlnLeu His Arg Thr Gly Ser Glu Ile His Pro Ala Asp Gly Tyr 130 135 140 AspGly Pro Gln Gly Gly Gly Tyr Pro Glu Pro Gln Gly Ala Arg Asp 145 150 155160 Ile Tyr Ser Tyr His Ile Lys Gly Thr Ser Thr Lys Thr Lys Ile Asn 165170 175 Thr Val Pro Gln Ala Pro Phe Ser Asp Arg Trp Leu Lys Glu Asn Ala180 185 190 Gly Ala Ala Ser Gly Phe Leu Ser Arg Ala Asp Glu Ala Gly LysLeu 195 200 205 Ile Trp Glu Asn Asp Pro Asp Lys Asn Trp Arg Ala Asn ArgMet Asp 210 215 220 Asp Ile Arg Gly Ile Val Gln Gly Ala Val Asn Pro PheLeu Thr Gly 225 230 235 240 Phe Gln Gly Leu Gly Val Gly Ala Ile Thr AspSer Ala Val Ser Pro 245 250 255 Val Thr Tyr Ala Ala Ala Arg Lys Thr LeuGln Gly Ile His Asn Leu 260 265 270 Gly Asn Leu Ser Pro Glu Ala Gln LeuAla Ala Ala Ser Leu Leu Gln 275 280 285 Asp Ser Ala Phe Ala Val Lys AspGly Ile Asn Ser Ala Arg Gln Trp 290 295 300 Ala Asp Ala His Pro Asn IleThr Ala Thr Ala Gln Thr Ala Leu Ala 305 310 315 320 Val Ala Glu Ala AlaGly Thr Val Trp Gly Gly Lys Lys Val Glu Leu 325 330 335 Asn Pro Thr LysTrp Asp Trp Val Lys Asn Thr Gly Tyr Glu Lys Pro 340 345 350 Ala Ala ArgPro Met Gln Thr Val Asp Gly Glu Met Ala Gly Lys Asn 355 360 365 Lys ProPro Lys Pro Ser Thr Gln Gln His Ser Thr His Ser Asp Asn 370 375 380 AsnIle Gly Leu Pro Ala Pro Tyr Val Lys Pro Asp Thr Ser Ile Ser 385 390 395400 Pro Thr Gly Thr Ile Gln Asp Arg Ile Arg Trp Thr Lys Ser Lys Phe 405410 415 Pro Thr Glu Lys Ser Leu Asn Gly His Phe Lys Ala His Gly Lys Glu420 425 430 Phe Gly Asp Ile Thr Ile Glu Asp Tyr Gln Lys Met Ala Ser AspLeu 435 440 445 Leu Ser Lys Gln Thr Ser Asp Lys Ile Leu Gly Tyr Gln ThrGlu His 450 455 460 Arg Arg Val Arg Tyr Asp Ile Asn Asn Asn Ile Tyr ValLeu Ala Asn 465 470 475 480 Pro Lys Thr Phe Lys Ile Lys Thr Met Phe LysPro Asn Leu Gly Lys 485 490 495 Glu Tyr Tyr Asp Gly Glu Phe Lys Lys AspMet Gly Asn 500 505

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of (a) a nucleic acid molecule comprising thenucleotide sequence depicted in SEQ ID NO: 1; (b) a nucleic acidmolecule comprising a nucleotide sequence having 95% sequence identityto SEQ ID NO:1 due to the degeneracy of the genetic code; and (c) anucleic acid molecule comprising a nucleotide sequence that hybridizesunder stringent hybridization conditions of 0.2×SSC, 0.1% SDS and 68° C.to the complement of SEQ ID NO: 1; wherein an open reading framecontained in the nucleic acid molecule encodes a protein from bacteriaof the genus Neisseria that mediates the adhesion of Neisseria cells tohuman cells.
 2. The isolated nucleic acid molecule according to claim 1,wherein the molecule originates from a pathogenic Neisseria species. 3.The isolated nucleic acid molecule according to claim 2, wherein theNeisseria species is Neisseria gonorrhoeae or Neisseria meningitidis. 4.A vector comprising a nucleic acid molecule according to claim
 1. 5. Ahost cell comprising the nucleic acid molecule according to claim
 1. 6.An isolated fragment of the nucleic acid molecule according to claim 1encoding a lipoprotein or biologically active fragment of saidlipoprotein that mediates adhesion of Neisseria cells to human cellsfrom a bacteria of the genus Neisseria selected from the groupconsisting of (a) a nucleic acid molecule encoding a protein having theamino acid sequence as depicted in SEQ ID NO:7; (b) a nucleic acidmolecule encoding a protein having the amino acid sequence depicted inSEQ ID NO:7 from amino acid residue 19 to amino acid residue 320; (c) anucleic acid molecule comprising a nucleotide sequence having 95%sequence identity to (i) a nucleotide sequence encoding a proteincomprising SEQ ID NO:7, and (ii) a nucleotide sequence encoding aprotein having the amino acid sequence depicted in SEQ ID NO:7 fromamino acid residue 19 to amino acid residue 320; and (d) a nucleic acidmolecule comprising a nucleotide sequence that hybridizes understringent hybridization conditions of 0.2×SSC, 0.1% SDS and 68° C. to(i) the complement of a nucleotide sequence encoding a proteincomprising SEQ ID NO:7, (ii) the complement of a nucleotide sequenceencoding a protein having the amino acid sequence depicted in SEQ IDNO:7 from amino acid residue 19 to amino acid residue
 320. 7. Thenucleic acid molecule according to claim 6, wherein the lipoprotein orbiologically active fragment of said lipoprotein has the ability toadhere to human cells.
 8. The nucleic acid molecule according to claim7, wherein the protein or biologically active fragment possesses theability to adhere to human cells in complexes with the protien PilC. 9.A vector comprising a nucleic acid molecule according to claim
 6. 10.The vector according to claim 9, wherein the nucleic acid molecule isoperatively linked to at least one regulatory DNA element allowing theexpression of said nucleic acid in a prokaryotic or a eukaryotic cell.11. A host cell comprising a nucleic acid molecule according to claim 6.